A variety of virus capsid proteins have the intrinsic ability to self-assemble into highly organized particles. By using recombinant DNA techniques, capsid proteins can be recombinantly produced from different hosts such as mammalian cells, insect cells, yeast and E. coli. Often, the produced capsid proteins can self-assemble into particles in the hosts that closely resemble virions. The resulted particles are called virus-like particles (VLPs). Because of lacking viral genome, VLPs are nonreplicating and noninfectious (1-14).
There are numerous documented research work and granted patents in the area of using VLPs prepared from virus capsid proteins as vaccines or using VLPs as antigen carriers or antigen delivery systems (or vehicles) to carry desired epitopes or antigens, in efforts to enhance the immunogenicity of the carried epitopes or antigens, and to prime in vivo class I-restricted cytotoxic responses (1-14, 62-68). Most of the antigen-delivery systems are formulated in particles or particulate in nature. A variety of materials, including lipids, proteins, polysaccharides, polyacrylic substances or organic acids are formulated into particles to serve as antigen delivery systems. Among them, capsid proteins formed virus-like-particles (VLPs) represent prime candidates as antigen carriers for the delivery of heterologous antigens for other diseases because of the ideal size of their particles, simplicity and the ability to induce desirable type of immune response (62-68). Also, the relative immunogenicity of different particulate antigen-delivery systems can be very different, capsid protein based particulate antigen-delivery systems may be more immunogenic than other particulate antigen-delivery systems (62-68, 109).
There is no doubt that VLPs can be expressed abundantly in a variety of expression systems by recombinant DNA techniques. There are very little doubts to the prophylactic or therapeutic potentials of using VLPs as vaccines or using them as antigen carriers for eliciting enhanced immune responses, particularly cell-mediated immune response against carried antigens or epitopes. Due to their particulate nature, VLPs usually can be purified in particles by methods such as salt precipitation with ammonium sulfate, density gradient centrifugation, and gel filtration. However, to use this technology to produce medicines, in particular for use in humans, there are still unsolved problems related to the economically and reproducibly preparing intact homogeneous particles from expression host systems with well defined compositions able to withstand long-term storage (8).
When produced by recombinant DNA technology, VLPs like many other recombinant proteins will be contaminated with host proteins, lipids, nucleic acids et al. These contaminations have to be removed to very low levels to meet the requirements for medical application. However, the removal of the contaminations from VLPs is complicated due to the fact that when VLPs are expressed and assembled in the expression systems, the host proteins and lipids can be incorporated into the VLPs and host nucleic acids can be packaged into the VLPs (15-20). Purification of whole VLPs will not be able to remove these incorporated or packaged contaminations. More ever, VLPs are super-molecular structures with molecular weight normally exceeds 10.00 Kd, Possibly due to the poor mass transfer in chromatographic processes because of the VLPs' massive sizes compare to monomer proteins or other small molecules, when the separations are conducted by using absorbent resins, the binding, elution and fractionation are not as effective and efficient as smaller molecules.
The importance of being able to purifying totally dissembled capsid proteins are noted in U.S. Pat. No. 6,962,777 and others (8, 21). VLPs' assembly requires correctly-folded capsid proteins to start with. Under non-denaturing conditions, the in vitro method for the quantitative disassembly and subsequent reassembly of VLPs is highly specific for each individual capsid protein and U.S. Pat. No. 6,962,777 might be the only published work dealt with this issue with VLPs prepared from human papillomavirus (HPV) L1 major capsid protein. Many factors significant for VLPs formation and stability have not been well elucidated. It is generally known that VLPs' disassembly and assembly can be affected by numerous factors. For example, pH, ionic strength, post-translational modifications of viral capsid proteins, disulfide bonds, and divalent cation bonding. To make this issue even more complicated is that the VLPs' disassembly and assembly often require chaperones participation and for some VLPs formation, certain specific structure nucleic acids are required (8, 21-36). Thus, there are numerous interrelated factors which may affect capsid stability, assembly and disassembly in vitro, which vary widely even for related viruses. Further more, the tendency of forming aggregates by partially dissembled or totally dissembled capsid proteins is another major obstacles in the process of producing homogenous and stable VLPs effectively and efficiently in vitro (8).
To simply dissemble VLPs, high concentration of Chaotropic agents such as urea or guanidine hydrochloride (Gu.HCl) can be used, as these agents will disrupt non-covalent forces such as hydrogen bonds, Van der Waals interactions, and the hydrophobic interactions in the capsid proteins, and the disulfide bonds in capsid proteins can be disrupted simply by reducing agents or oxidative sulfitolysis process. If just for producing pure capsid proteins, then to subject VLPs in high concentration of Gu.HCl and urea, plus necessary reagents to disrupt disulfide bonds in the purification process would be advantageous because of the following reasons. (1) Capsid proteins are much less likely to form aggregates in high concentration of urea or Gu.HCl, so the purification process can be much more efficient and scalable; (2) High concentration of urea or Gu.HCl can weak the interactions (hydrogen bonding, Van der Waals interactions, and the hydrophobic effect) between capsid proteins and contaminations; so the purification process can be much more effective in terms at removing contaminations; (3) VLPs are disintegrated in the high concentration of urea or Gu.HCl, so the capsid proteins exhibit more homogenous properties in the purification process; (4) disintegrated VLPs are much more likely to release or expose incorporated or packaged contaminations to the purification forces for removing them. However, the chaotropic agents such as urea or Gu.HCl are also strong protein denaturants, proteins are denatured after the treatment with high concentration of urea or Gu.HCl, and there is still lack of knowledge on how to correctly refold denatured capsid proteins. If denatured capsid proteins are not correctly refolded, they often form aggregates instead of self-assemble into VLPs (8, 21).
A host's immune responses or immune tolerance to a virus may also pose problems for using authentic VLPs as antigen carriers. Capsid proteins formed VLPs represent prime candidates as antigen carriers for the delivery of heterologous antigens because of the ideal size of their particles, simplicity and the ability to induce desirable type of immune response (62-68). However, the host might have been infected with the viruses, and the infections may result the host developing immune responses to the VLPs prepared from the same viruses or close related viruses; or the infection may result host developing immune tolerance to the VLPs prepared from the same viruses or close related viruses. In either case, the effectiveness of using VLPs as antigen carriers in such host will be greatly reduced.
It can be advantages if capsid proteins with self-assembly ability can be utilized to prepare particles (macro-molecular structures) with different morphology from the authentic VLPs as antigen carriers. Because of the differences of the morphology, the macro-molecular structures will display or expose a different set of epitope peptides compare to authentic VLPs, and may have the following advantages over authentic VLPs: (1) pre-existing immunity to authentic VLPs might be circumvented by the use of macro-molecular structures with different morphology; (2) existing immune tolerance to authentic capsid proteins might be circumvented by the use of the macro-molecular structures with different morphology; (3) the use of macro-molecular structures with different morphology might circumvent the problem of interference with commercial anti-capsid protein assays.
Therefore, there exists a need in the art for a general method, which would conduct the purification of recombinantly expressed capsid proteins in one or more steps in high concentration of chaotropic agents (denaturing conditions) plus necessary agents to disrupt disulfide bonds, then refold and reassemble of purified homogenous capsid proteins. There also exists a need in the art to utilize capsid proteins to prepare particles (macro-molecular structures) morphologically different from authentic VLPs as antigen carriers for circumventing pre-existing immune responses to the VLPs or existing immune tolerance to the VLPs.